21. Description: Mutations in ras alleles occur at a high frequency in all human tumors, which emphasizes the importance of ras in cellular proliferation. Because of a similar prevalence of mutations in ras in both benign and malignant thyroid follicular neoplasms, it is thought that ras oncogenes are an early step in thyroid tumorigenesis. To investigate the complex events initiated after ras activation, we have developed PCCL3 rat thyroid cells expressing a valine 12 ras mutant under control of a doxycycline-inducible promoter. We find that after a four day period after doxycycline-inducible promoter. We find that after four day period after doxycycline induction of ras in PCCL3 cells, there is a marked increase in apoptosis, as determined by progressive cell detachment and DNA fragmentation. We believe that a growth promoting oncogene such as ras may induce cells to undergo apoptosis as an attempt to restrain unregulated cell proliferation and to maintain normal tissue homeostasis. We propose that in order for thyroid cells to become tumorigenic, they must acquire additional defects that disable the apoptotic. The aim of this proposal is to address this hypothesis by exploring the pathways downstream of ras that are involved in the activation of apoptosis and how these interact with survival factors. In specific aim 1, we will determine the conditions that either factor or interfere with ras-induced apoptosis in rat thyroid PCCL3 cells. Lack of growth factors or serum factors of serum deprivation as well as Bcl-2 over-expression will be explored. In specific aim 2, the signaling pathways utilized by mutant ras to activate apoptosis will be determined through the use of activated and dominant negative variant of MAP kinase, JN kinase, and p38MAP kinase. By investigating the pathways downstream of H-Ras v12 that are involved in activating apoptosis, much knowledge will be gained into the mechanisms of how thyroid tumors are initiated.